http://www.paper.edu.cn PO Box 2345, Beijing 100023, China World J Gastroenterol 2004;10(19):2836-2841 Fax: +86-10-85381893 World Journal of Gastroenterology E-mail: [email protected] www.wjgnet.com Copyright © 2004 by The WJG Press ISSN 1007-9327 • BASIC RESEARCH • Tegaserod inhibits noxious rectal distention induced responses and limbic system c-Fos expression in rats with visceral hypersensitivity Hong-Mei Jiao, Peng-Yan Xie Hong-Mei Jiao, Peng-Yan Xie, Department of Gastroenterology, INTRODUCTION First Hospital of Peking University, Beijing 100034, China Correspondence to: Peng-Yan Xie, Department of Gastroenterology, Irritable bowel syndrome (IBS) is a common disorder First Hospital of Peking University, Beijing 100034, characterized by abdominal pain and altered bowel habits, China. [email protected] consisting of constipation, diarrhea, or both. Several Telephone: +86-10-66551122 Ext. 2581 pathophysiological mechanisms have been suggested to play Received: 2004-03-15 Accepted: 2004-04-17 a role in the genesis of symptoms in patients with IBS, among others visceral hypersensitivity, autonomic nervous system dysregulation[1], alterations of gastrointestinal (GI) motility[2], and abnormalities in neurotransmitter systems[3]. It has been Abstract shown that at least a subgroup of IBS patients shows a AIM: To examine the effects of tegaserod, a serotonin hyperalgesic response to visceral stimuli, and discomfort in (5-HT) 4 receptor partial agonist, on abdominal withdrawal response to colorectal balloon distension under experimental reflex (AWR) to rectal distention (RD) and c-Fos expression conditions[3,4]. Abnormalities which upregulate afferent in limbic system. (sensory) signal intensity anywhere in the “brain-gut axis” could induce visceral hypersensitivity[5]. METHODS: Neonatal Sprague-Dawley rats randomly It has been shown in experimental rats that rectal distention received colonic irritation by acetic acid from postnatal day is a non-invasive, reproducible visceral stimulus, which can induce 8 to d 21 as a visceral hypersensitive model (group H) or by a range of pseudoaffective responses, including vasomotor, intrarectal saline as a control group (group C). When they visceromotor, and respiratory responses[3]. Abdominal withdrawal became adults, rectal distention (RD) was performed by a reflex (AWR) is an involuntary motor reflex similar to the balloon (6F; Fogarty arterial embolectomy catheter; length, visceromotor reflex[6]. Intestinal distention can be considered 20 mm; diameter, 2 mm) which was rapidly inflated with to as an appropriate stimulus for studies of visceral nociception[7]. increasing volumes of saline (0.4, 0.8 and 1.2 mL) for 20 s In previous studies, it has been shown that noxious distension at five-minute intervals. Five subgroups of group H (H-saline, of hollow viscera induces a specific pattern of c-Fos expression H-vehicle, H-Teg0.1, H-Teg0.3 and H-Teg1.0) were injected in rat limbic brain structures[3,8], which involved in higher randomly with saline, vehicle (1-methyl-2-thpyrrolidone) or cognitive functions (i.e. emotion, memory, motivation) and led tegaserod at doses of 0.1, 0.3 and 1.0 mg/kg ip, respectively. to the perception of visceral pain[9]. Induction of c-Fos expression Two subgroups of group C (C-Saline and C-Teg1.0) were is a well established marker of neuronal activation, and injected with saline or tegaserod (1.0 mg/kg) ip. RD was immunohistological detection of c-Fos-like immunoreactivity performed 10 min after injection, AWR was recorded and allows a mapping of activated brain nuclei on a single cell level[3]. c-Fos expression in limbic system was analyzed quantitatively Serotonin (5-HT) is thought to play a role in visceral by immunohistochemistry. nociceptive mechanisms. There is considerable evidence that serotonin is involved in the regulation of motility and sensation RESULTS: Compared to saline, tegaserod significantly in the gut[3]. In animal studies, tegaserod was reported to inhibit inhibited AWR in group H (0.4 mL: from 2.0 to 0.5; 0.8 mL: abdominal contraction response to noxious intestinal distention[10]. from 3.5 to 1.5; 1.2 mL: from 4.0 to 3.0, P<0.01), but had Tegaserod, a 5-HT4 receptor partial agonist, could relieve no significant effect on group C. Tegaserod dose-dependently symptoms in irritable bowel syndrome patients with abdominal attenuated the number of c-Fos positive neurons in limbic pain, bloating and constipation[11]. However, little is known structures, anterior cingulate cortex (ACC) showed the about the effect of tegaserod on neuronal activity in limbic greatest attenuation. In group H, tegaserod (1.0 mg/kg) structures at noxious rectal distention. Therefore, in the present resulted in a significant overall decrease to 57% of H-saline study, we established a rat model to investigate the role of 5- (283±41 vs 162±16, P<0.01), in ACC to 42% of H-saline HT4 receptors in mediating activation of limbic structures at (72±10 vs 31±8, P<0.01). In group C, tegaserod (1.0 mg/kg) rectal distention, as assessed by c-Fos expression. We aimed resulted in an overall decrease to 77% of C-saline (214±13 to establish a mechanism of the action of 5-HT4 receptors vs 164±22, P<0.01), in ACC to 65% of C-saline (48±8 vs specific to visceral nociceptive neurotransmission. 31±7, P<0.01). MATERIALS AND METHODS CONCLUSION: Tegaserod inhibits the response to rectal distention in rats with visceral hypersensitivity and dose- Animals dependently attenuates c-Fos expression in limbic system, Experiments were performed using Sprague-Dawley rats especially in anterior cingulate cortex. obtained as preweanling neonates (younger than 8 d) from the Animal Center in the First Hospital of Peking University. Rats Jiao HM, Xie PY. Tegaserod inhibits noxious rectal distention were housed in plastic cages containing corn chip bedding induced responses and limbic system c-Fos expression in rats and maintained on a 12:12-h light-dark cycle (lights on at 7 with visceral hypersensitivity. World J Gastroenterol 2004; AM) at 22 to 23 and in 60-65% humidity. The irritation 10(19): 2836-2841 procedure and the experimental testing were conducted during http://www.wjgnet.com/1007-9327/10/2836.asp the light component of the cycle. The neonates were housed 转载 中国科技论文在线 http://www.paper.edu.cn Jiao HM et al. Tegaserod on visceral hypersensitivity 2837 12 in a cage with their mothers until they were 25 d old. Mothers abdomen; 4, body arching and lifting of pelvic structures. The had access to food and water ad libitum. After separation, the rats were given RD for 20 s every 5 min. To achieve an accurate rats were housed 4 in a cage with access to food and water ad measure, distensions were repeated 5 times for each volume. libitum. The animals were deprived of food but water 18 h The data for each animal were averaged for analysis. The results before rectal distention (RD). Animal care and experimental obtained were compared among groups. A change in the procedures were followed institutional ethics guidelines and magnitude of an evoked response indicated a change in visceral conformed to the requirements of the State Authority for pain processing. Animal Research Conduct. Colon stimuli [12] Weight Colon stimulation consisted of graded RD produced by inflating Each rat was weighed every 3 d from days 9 to 40. a balloon inside the rectum. The balloon, 2 cm in length and 2 mm in diameter (6F, Fogarty arterial embolectomy catheter, Colon irritation Baxter, USA), was carefully inserted intrarectally and fixed at a Neonatal Sprague-Dawley rats (8 d old) were divided into 2 distance of 1 cm with an adhesive tape at the tail of the rat. groups (group C: control and group H: hypersensitivity) Distension was produced by rapidly inflating the balloon to undergoing different treatments. Forty-eight rats in group H the desired volumes with saline (0.4, 0.8 or 1.2 mL) for 20 s at 5-min received intracolonic injections of 5 mL/L acetic acid (0.5 mL) intervals. Before they were used, the balloons were blown up daily between the ages of 8 and 21 d. Acetic acid was injected and left overnight so the latex stretched and the balloons into the colon via the PE90 tube inserted to 2 cm from the anus. became compliant. Tegaserod (0.1, 0.3 or 1.0 mg/kg) or saline or Twenty-four rats in group C received intracolonic injections of vehicle was administered 10 min prior to RD. Only a single dose 9 g/L saline (0.5 mL) daily between the ages of 8 and 21 d[12]. was tested in each animal. [15] Drug administration protocol c-Fos immunohistochemistry Because tegaserod (HTF 919; Novartis Pharma AG, Basel, Within 30 min following the end of the distention procedure, Switzerland) is poorly soluble in water, the fractions were made the animals were deeply anesthetized with an overdose of up using 0.1 mL of 1-methyl-2-pyrrolidinone (vehicle)[10]. After sodium pentobarbitone (60 mg/kg intraperitoneally) and then perfused through the ascending aorta with saline (9 g/L), dissolved in the vehicle, distilled water was added to make followed by 500 mL of cold 0.1 mol/L phosphate buffer (PB, 4 ) the solution up to 0.5 mL. According to the drugs injected containing 40 g/L paraformaldehyde (pH 7.4). The brain was intraperitoneally 10 min before RD, 48 rats in group H were immediately removed and postfixed in the same fixative at 4 divided into 6 subgroups (H0, H-saline, H-vehicle, H-Teg0.1, overnight, and then placed in 300 g/L sucrose with 0.1 mol/L H-Teg0.3, and H-Teg1.0), and 24 rats in group C were divided PB for 72 h at 4 .